The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs). GPCRs are proteins that span the membrane of a cell and relay the information provided by numerous ligands, e.g. hormones and neurotransmitters, into intracellular signalling pathways. GPCRs are thus the targets of many clinically important drugs, with approximately half of all current prescription drugs acting through GPCRs (Drews J (1996) Genomic sciences and the medicine of tomorrow. Nat Biotechnol 14: 1516-1518). Examples of GPCRs are many and include beta-2 adrenergic receptor (β2-AR), Frizzled 4 (Fz4), V2-vasopressin receptor (V2R), V1a vasopressin receptor (V1aR), δ-opioid receptor (δ-OR), platelet-activating factor receptor (PAFR), CC chemokine receptor type 5 (CCR5), and angiotensin receptor type 1a (AT1aR).
GRCRs relay the information encoded by the ligand (e.g. hormones and neurotransmitters) through the activation of G proteins and intracellular effector molecules. G proteins are heterotrimeric proteins, consisting of an alpha, a beta, and a gamma subunit. The three G-subunits are non-covalently bound together and the G protein as a whole binds to the inside surface of the cell membrane and associates with the GPCR. Starting in such conformation, the G-alpha subunit is complexed to GDP (guanosine diphosphate). When a ligand binds to a domain of the GPCR accessible from the outside of the cell membrane, a conformational change in the GPCR occurs, which in turn prompts the exchange of the GDP for a molecule of guanosine triphosphoate (GTP) on the G-alpha subunit, and activates the G-protein. The G-protein's α subunit, together with the bound GTP, can then dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly, depending on the α subunit type (e.g. Gαs, Gαi/o, Gαq/11, Gα12/13).
In order to turn off this response by GPCRs to stimulus, or adapt to a persistent stimulus, the activated GPCRs are inactivated. This inactivation may be achieved, in part, by the binding of a soluble protein, β-arrestin (β-arr), which uncouples the receptor from the downstream G protein after the receptor is phosphorylated by a G protein-coupled receptor kinase (GRK). More specifically, through their binding to agonist-occupied, GRK-phosphorylated receptors, β-arrs prevent further coupling to G proteins and promote GPCR endocytosis, thus leading to decreased signalling efficacy.
Despite our growing understanding of the diversity in GPCR signaling mechanisms, drug efficacy is often defined only in terms of the regulation of the classical G protein signaling. Within this framework, agonists are defined as drugs that stabilize an active receptor conformation that induces G protein activation, whereas inverse agonists favor an inactive receptor state that reduces spontaneous G protein signaling. The question arises as to whether this paradigm may be transferred to drug effects generated through the formation of metastable complexes involving scaffolding proteins such as β-arr. Because all studies describing β-arr-mediated MAPK signalling have concentrated on agonist drugs, little is known of how ligands that are commonly classified as inverse agonists may regulate the scaffold assembly that is crucial for such signalling.
In one study (Azzi et al, 2003), this question was addressed by assessing whether β-adrenergic receptor (β2AR) and V2 vasopressin receptor (V2R) ligands with proven inverse efficacy on adenylyl cyclase (AC) activity could also regulate MAPK activation via receptor-mediated scaffold formation. It was found that, despite being inverse agonists in the AC pathway, the β2AR (ICI118551 and propranolol) and V2R(SR121463A) induced the recruitment of β-arr leading to the activation of the ERK cascade. Such observations indicate that the same drug acting on a unique receptor can have opposite efficacies depending on the signaling pathway considered.
The above study relied on the use of a bimolecular bioluminescence resonance energy transfer (BRET) assay. It was used to assess β-arrestin recruitment to β2AR or V2R. Fusion proteins consisting of GFP10 variant (GFP) covalently attached to the carboxyl tail of the receptor of interest (β2AR-GFP; V2R-GFP) were co-expressed with β-arrestin 2 fused at its carboxyl terminus to Rluc (β-arrestin-Rluc). After incubation of the transfected cells with different ligands, coelenterazine 400a (Perkin-Elmer, Wellesley, Mass., USA) was added and readings were collected using a modified top-count apparatus (BRETCount, Packard) that allows the sequential integration of the signals detected at 370-450 nm and 500-530 nm. The BRET signal was determined by calculating the ratio of the light emitted by the Receptor-GFP (500-530 nm) over the light emitted by the β-arrestin2-Rluc (370-450 nm). The values were corrected by subtracting the background signal detected when the β-arrestin2-Rluc construct was expressed alone.
While the results elicited from the above study were instructive, a necessary feature involved the construction of fusion proteins that included the receptors of interest. Ideally, a method could be devised in which receptor activation might be observed without first having to modify the receptors that are to be studied. Other features of such a method that would make it highly desirable for research and development endeavors include the following: (1) a high level of sensitivity; (2) an ability to provide quantitative results; (3) adaptability for use in large scale screening analyses; (4) an assay that requires the expression of a single recombinant construct; and (5) a biosensor based on an intramolecular RET signal.
Resonance energy transfer (abbreviated RET, and also referred to as Förster resonance energy transfer), is a mechanism describing energy transfer between two chromophores, having overlapping emission/absoprtion spectra. When the two chromophores (the “donor” and the “acceptor”), are within 10-100 Å of one another and their transition dipoles are appropriately oriented, the donor chromophore is able to transfer its excited-state energy to the acceptor chromophore through nonradiative dipole-dipole coupling. When both chromophores are fluorescent, the term typically used is “fluorescence resonance energy transfer” (abbreviated FRET). In bioluminescence resonance energy transfer (BRET), the donor chromophore of the RET pair, rather than being a fluorophore, is a bioluminescent molecule, typically luciferase. In the presence of a substrate, bioluminescence from the donor excites the acceptor fluorophore through the same Förster resonance energy transfer mechanism described above (Xu, Y. et al., PNAS, 96:151-156 (1999)).
There is a need for a simpler method to measure receptor activity in living cells. The present invention seeks to meet this and related needs.